PROJECT SUMMARY Despite decades of work, there has been little success in engineering scaffolds that can successfully restore the enthesis, the tissue smoothly transfers muscle-generated force from tendon to bone. This region is prone to failure from excessive mechanical loading and in many cases the interface cannot be surgically reestablished due to the complexity and low cellularity of the enthesis. A reason engineered scaffolds lack the ability to restore the damaged enthesis is that the design predominantly mimics the architecture and composition of the mature tissue. What is rarely taken into consideration in scaffold design is that tissues undergo extensive ECM remodeling during development, which plays a significant role in directing cellular behavior in the formation of the mature tissue. Researchers have been unable to capitalize on these instructive cues for scaffold design due to the limited knowledge regarding the composition, turnover, organization and mechanical properties of developing musculoskeletal tissues. Our long-term objective is to create scaffolds that can biomechanically direct cells to rebuild damaged tissues; therefore, it is critical to identify how this is accomplished in vivo. To achieve our objective, we need to first address the following questions: 1) What are the dynamics of ECM expression over the course of enthesis formation? 2) How are these components organized in 3D? 3) How does this organization influence the mechanical environment? 4) How does mechanical loading regulate enthesis assembly? To directly quantify ECM protein incorporation into the matrix of developing tendon, enthesis and cartilage, we will label tissues at various stages of murine development with non-canonical amino acids (ncAAs). The bioorthogonal handles on the ncAAs enable the identification and localization of newly synthesized proteins using click chemistry. To see how individual ECM components are spatially distributed with respect to cells in the developing enthesis, we will use optical clearing methods to visualize murine tissues containing fluorescently labeled tendon and cartilage progenitors. Using confocal microscopy and 3D image processing algorithms, we will characterize how morphology at the intracellular, cellular and tissue scale change due to development and embryonic motility. To test the hypothesis that the stiffness across the enthesis will develop a steeper gradient upon the onset of embryonic and postnatal motility, we will utilize our novel atomic force microscopy method that can measure the stiffness of cells and ECM within viable tissues. This hypothesis will be directly tested by employing the mdg model of muscular dysgenesis, a mouse line in which skeletal muscle contractility is inhibited during embryogenesis. By correlating the mechanical properties with the compositional and structural characterization, we expect to identify a set of scaffold parameters that will promote cellular behaviors necessary for enthesis regeneration.